Andy Kravetz (akravetz at MO.NET) wrote:
: why. Here are some facts and maybe y'all can help me. The insert was PCR
: amplified to have the two restriction sites that I need, Nco1 and EcoR1.
As the other people who responded have mentioned, make sure the
restriction sites are far enough away from the ends of the fragment to
get good cutting.
: water bath that can hold 16C in the cold room. The conc. of the two are
: Insert 90ng/ul and Vector 500ng/ul.
I think your concentration of insert is kind of low. I would start with
more insert. Since you are PCR amplifying the insert it shouldn't be
hard to get more of it. When I have trouble cloning things I always
increase the amount of insert I use in the ligation. That always
works. Sometimes you just have to bash the dam things in there.
Hope this helps,
George